key: cord-0331418-ptd0nccb
authors: Mohsenpour, Reza; Sabet, Saeed Shafiei
title: Experimental sound exposure modifies swimming activities and increases food handling error in adult zebrafish (Danio rerio)
date: 2021-12-17
journal: bioRxiv
DOI: 10.1101/2021.12.01.470707
sha: f41a7f8089fd9e60ae15ce03b48276df933c3019
doc_id: 331418
cord_uid: ptd0nccb

Anthropogenic sound is globally increasing and is recognized as a source of environmental pollution in terrestrial and aquatic habitats. Sound is an important sensory stimulus for aquatic organisms and it may cause fluctuations in stress-related physiological indices and in a broader extent induce behavioural effects in a range of marine and freshwater fishes. However sound exposure may also induce changes in swimming activities, feed efficiency and productivity of available food sources in fish. Here, we experimentally tested sound effects on swimming activities and foraging performance in thirty adult Zebrafish (Danio rerio) individually in captivity. We used adult zebrafish and water flea (Daphnia magna) as model predator and prey, respectively. In terms of acoustic stimuli, we used four sound treatments with different temporal patterns (all in the same frequency range and moderate exposure level). Our results constitute strong evidence for sound-related effects on zebrafish behaviour. All sound treatments led to a significant increase in the number of startle response, brief and prolonged swimming speed for zebrafish (P<0.05). We found partially brief and prolonged sound effects on the spatial distribution of zebrafish; Although there were not any significant sound-related changes for horizontal spatial displacement in all treatments (P>0.05), zebrafish swam significantly more in the lower layer of the tank except for irregular intermittent 1:1-7 in brief sound exposure (P<0.05). The results of foraging performance showed that food discrimination error unaffected by sound treatments and was low for the zebrafish (P>0.05). However, food handling error was affected by sound treatments; all treatments induced a significant rise in handling error (P<0.001). This study highlights the impact of sound on zebrafish swimming activities, and that more feeding bouts are needed to consume the same number of food items increasing energy demand under noisy conditions.

Nowadays, due to the increase in human activities and the advancement of technology globally 33 since the Industrial Revolution, the living environment has undergone extensive changes 34 (Normandeau Associates, 2012). These environmental changes can affect the planet and living 35 organisms, and that can be a major threat to the biodiversity inhabit Earth (Kunc et al., 2016) . 36 The rapid growth of these changes poses many environmental challenges (Tuomainen and 37 Candolin, 2011) in both terrestrial and aquatic habitats. One of the main sources of 38 environmental pollution which may also can be recognized as an environmental stress stimulus 39 is anthropogenic sound that in addition to affecting terrestrial animals, also have many (Tyack, 1998; Popper and Hastings, 2009; Slabbekoorn et al., 2010) . 59 Behavioural effects are the most likely to occur and thus play as a stress driver (Smith et al., of prey items and predatory species. Such changes in turn may increase foraging energy 97 demand and the amount of time allocated by fish to foraging which, subsequently induce a 98 number of major changes such as affect food searching, discriminating and handling. 99 In general, Danio rerio is known as a model fish species in behavioural studies and responding 100 to environmental conditions (Cachat et al., 2010; Egan et al., 2009; Whitfield, 2002) . Zebrafish 101 is a member of the Cypriniformes order and acclimates well in captivity (Detrich et al., 2011) 102 and naturally lives in the tropical freshwater (Spence et al., 2008) . Zebrafish have specialized 103 hearing structures, the Weberian ossicles, with an auditory system that has homologies to the 104 mammalian auditory system (Weber, 1820; Alexander, 1964; Whitfield, 2002) . 105 Progress in the field of behavioural biology over conservation ecology and findings about the 106 potential impacts pollutants on organisms is also to a large extent linked with the study of 107 invertebrates. Daphnia is a small crustacean and inhabits in open and light waters, also they are 108 an important part of the food web in freshwater habitats and inhabits many types of shallow 109 water bodies (Ebert, 2005 ; Parejko and Dodson, 1991; Reynolds, 2011) . 110 In the present study, as a follow up of our recent study (Shafiei Sabet et al., 20215), we 111 experimentally tested the hypothesis that whether experimental sound exposure ensonified by 112 an underwater speaker affect the general swimming activities and foraging behaviour of 113 zebrafish as predator upon water flea as prey item under laboratory conditions. Comparing to 114 Shafiei Sabet er al., 2015 that we used an in-ear speaker, in this study we adopted an underwater 115 speaker to increase sound pressure levels and potential gradient of it in the arena. We also 116 extended the time of behavioural analysis to explore more long-term effects of sound on the 117 zebrafish. Our specific goals were: firstly, to assess the effect of experimental sound exposure 118 on zebrafish activities including swimming speed and spatial displacement. Secondly, to 119 estimate whether the temporal pattern of sound exposure matters and affects differently 120 zebrafish behaviour. And thirdly, to verify our recent laboratory-based findings of sound 121 impacts on zebrafish swimming activity and foraging behaviour. 

This study was performed in the ornamental fish breeding facility center at University of 124 Guilan, Sowmeh Sara, Iran (37°17ʹ39ʺN, 49°19ʹ55ʺE), using an aquarium with dimensions of 125 50 ×15 ×20 cm in the period of 10:00 to 14:00 every day. Zebrafish (~ age of 45 days old and 126 of the wild-type, short-fin variety weight (± s.d.) of 1.23 ± 0.02 g) were obtained from an 127 ornamental fish breeding shop in Sowmeh Sara, Iran. Zebrafish were stored in a stock glass 128 tank with dimensions 50×30×40 cm for two weeks and adapted to environmental conditions to 129 reduce possible stress and hormonal changes due to transportation, captivity conditions and 130 animal welfare issues (Deakin et al., 2019 including the first treatment as control treatment in which the fish were exposed to ambient 137 noise (AN), regular intermittent sound (IN) with fast pulse rate (1:1) ( Fig. 1 (a) ), regular 138 intermittent sound with slow pulse rate (1:4) ( Fig. 1 (b) ), irregular intermittent sound (1:1-7) 139 ( Fig. 1 (c) ), and continuous sound (CS) ( Fig. 1 (d) ). All three intermittent treatments include 140 one second of sound, but the difference between these sound treatments is the intervals between 141 these sounds (silence time) (See also Shafiei Sabet et al., 2015) . The sound treatments were 142 generated and modified using Audacity® software (2.3.1) at the sound frequency that can be 

The experimental tank with dimensions of 50×20×15 cm with black background was prepared 150 to improve the contrast between Daphnia and fish in the video file. The zebrafish swimming 151 activities were filmed by a video camera (Panasonic HC-V180 Full HD) at a distance of 50 cm 152 from in front of the test tank. Sound treatments were played back as stereo WAV files using a 153 Laptop (Sony Vaio SVF1421A4E) connected to an underwater speaker (custom-build speaker, 154 30 W, 10 Hz-10 KHz). In this experiment, a divider plate was placed transversely in the tank 155 and the tank length was halved (25×20×15 cm) in order to increase the enclosure on the 156 zebrafish swimming environment and make the entire fish swimming space visible for video-sounds and reduce unwanted sounds, we placed and covered all walls at the entrance to the 161 experimental room and also inside walls using egg boxes. 162 The underwater speaker used in the experiment was placed horizontally on the other side of 163 the separator plate (See Fig. 2) . Then, each test was performed as follow: after ten minutes, the 164 sound treatment was played by a speaker and a sound player for 20 minutes, However, the food 165 item (Daphnia) and non-food item (Duckweed) were added to the experiment tank after ten 166 minutes from onset of each sound playback in all sound treatments with a 15-minute interval 167 between treatments and the fish was exposed to all five acoustic treatments and repeated the 168 next day for the next fish. The order of broadcast of sound treatments on a daily basis was 169 randomly balanced. We used a similar methodological approach previously described to The effect of sound on the foraging behaviour of zebrafish was investigated in such a way that 206 5 waterfleas (~3 mm) as a prey species (target) and 5 non-food substances as non-food item in 207 the same size as Daphnia (~ 3mm) was mixed in 25 ml beaker container and added gently to 208 the fish tank in the same manner for all treatments. The waterfleas were in the same sizes that 209 caught with plastic Pasteur pipettes to decrease damaging water, which is suitable for feeding Kolmogorov-Smirnov test and the homogeneity of the data by Levene test. Then, the presence 222 or absence of significant differences between the mean of the data was assessed by repeated 223 measures ANOVA analysis and using Tukey multi-range test. A HuynheFeldt correction was 224 performed when sphericity could not be assumed in the repeated measures ANOVA.

Bonferroni corrected post hoc tests were performed when ANOVA test results were significant. 226 The level of significance in this study was considered P<0.05. A custom-written acoustic 227 calibration script in R studio software (Version 1.1.456) was also used to evaluate sound 228 pressure levels and power spectral density that were played by the underwater speaker. However, all acoustic treatments showed a significant difference compared to the control 305 treatment (repeated measure ANOVA: F2.82,81.91=26.023, P≤0.001) but no significant 306 difference was observed between sound treatments (P>0.05) (Fig. 9b) . This means that with 307 the broadcast of acoustic treatments compared to the control treatment, there was a significant 308 handling error in food intake in the food. performance in zebrafish as all will be discussed further as follows in the next sections. Startle response by prey fish is a behavioural response to enhance survival rate in predator-353 prey relationships (Webb, 1986) . By hearing the sounds of predator fish and receiving sound 354 signals related to the attack, the prey fish starts swimming at high speed and explosively in the 355 opposite direction of the perceived sound in order to increase the success rate of escaping and 356 staying away from the predatory species. Sounds can affect the prey fish's decision-making 357 power against sound sources or danger, the way in which prey assesses risk (Dukas, 2004) , and The use of speakers in air to broadcast sound treatments leads to the production of more sound In order to understand the behavioural changes of zebrafish in response to acoustic stimuli, first 484 of all, it is very important to understand the mechanisms behind and that how the species detects 485 and processes, and how it behaviourally responds to sound .

Because well-documented studies already showed that the auditory system of fishes evolved 487 primarily to detect particle motion, many fishes are most sensitive to particle motion and they consists of both sound pressure and particle motion . In fact, all 491 fishes and all invertebrates primarily detect particle motion but not all detect sound pressure.

Only some of fishes, including the zebrafish, are sensitive to sound pressure as well as the 493 particle motion (Popper and Fay, 2011; Popper and Hawkins, 2018) . There are some studies 494 revealing directional hearing and sound source localization in fish under laboratory conditions 495 and in free sound fields. Schuijf (1975) proposed that the cod (Gadus morhua) are able to 496 detect sound directionality by monitoring the particle motion of the sound field, presumably 497 employing the directional orientation of the inner ear sensory cells (Dale, 1976) . Although, 498 Schuijf (1975) also concluded that to determine the direction of a sound source, the direction 499 of only particle motion may not be sufficient. It has already been shown that cod could 500 discriminate between signals coming towards the head as compared to those coming towards 501 the tail (Buwalda et al., 1983; Schuijf and Buwalda, 1975) . They argued that to eliminate any 502 remaining 180 0 ambiguities, directional hearing might involve both comparing the responses 503 of hear cells oriented in different directions and also analysis of the phase relationship between 504 the sound pressure and particle motion components (Schuijf, 1976) . 506 We did not mention the levels and direction of the particle motion that is generated within the 507 fish tank because of our restriction in acquiring the particle motion measurements devices and 508 the COVID-19 pandemic. Therefore, we believe it is premature to conclude that zebrafish 509 cannot localize sound source in our experimental set up. One might be because we know very 510 little about hearing in fishes only over 120 species of the more than 33000 known fish species 511 (Ladich and Fay, 2013) and that the empirical and theoretical work on sound source localization 512 and directional hearing in fishes have been contradictory and obscure for decades (Sisneros 513 and Rogers, 2016). Moreover, some explanations would be that practically because it is 514 difficult to monitor particle motion in fish tank, the lack of easily used and reasonably priced 515 instrumentation to measure particle motion, lack of sound exposure criteria for particle motion 516 and finally lack of particle motion measurement standards (Popper et al., 2014) . 517 Within an aquarium tank the levels of particle motion are often highest at the water surface, 518 and close to the tank walls, when an underwater loudspeaker is used (Jones et al., 2019) . In the present study, the parameters of fish foraging behaviour such as food discrimination 534 error and food handling error were examined. The results of this experiment showed that the 535 zebrafish did not show any significant difference in the food discrimination error when exposed 536 to sound treatments compared to the ambient treatment. Also, the results of food handling error 537 showed that all sound treatments showed a significant difference compared to the ambient 538 treatment, but these sound treatments did not show a significant difference compared to each In a study by Purser and Radford (2011) , they found that the broadcasting of sound treatments 544 significantly affected the foraging performance of stickleback and food discrimination error 545 and food handling error increased significantly compared to the ambient treatment and reduced 546 foraging performance, which in the food discrimination error, it is not consistent with the 547 results of the present experiment, and one of the reasons for this difference is the difference in 548 the physiology of fish and its diet. Also, the possible difference in the physiology of fish visual 549 sense can be one of the factors influencing these differences, but in the food handling error, a The authors declare that they have no known competing financial interests or personal 596 relationships that could have appeared to influence the work reported in this paper. Slabbekoorn for providing accessibility to acoustic measurement devices. We also thank Narjes

Karimi for helping us in reviewing the results and graphs. 

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Brief time (15 seconds before and 15 seconds 1100 exposed to sound). (b) Prolonged time (60 seconds before and 60 seconds exposed to sound). The blue color 1101 (#0000FE) indicates the 0-20 % of the fish in the tank. The aqua color (#01FFFF) indicates the 20-40 % of the 1102 fish in the tank. The lime color (#00FF01) indicates the 40-60 % of the fish in the tank. The yellow color 1103 (#FFFF01) indicates the 60-80 % of the fish in the tank. The red color (#FE0000) indicates the 80-100 % of the 1104 fish in the tank. The underwater speaker played back from the right tank

Food discrimination error; it described 1107 as the proportion of duckweed particles attacked relative to the total number of attacks to both duckweed particles 1108 and water fleas from the introduction of food items until the end of sound exposure in sequence for each zebrafish 1109 individual (Shafiei Sabet et al., 2015). (N=30, df=3.756, F=1.339, P=0.226) (b) Food handling error; It described 1110 as the proportion of the total of water fleas attacked that were missed or released again after initial grasping from 1111 onset of food introduction until the end of sound exposure in sequence for each zebrafish individual

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